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Judging a bacterium by its cover: immune cell–symbiont communication in the Hawaiian Bobtail squid/Vibrio fischeri symbiosis

By Sarah McAnulty (@SarahMackAttack), a doctoral candidate in the Nyholm lab (Department of Molecular and Cell Biology, University of Connecticut)

The first thing everyone tells me when they hear about my work is, “Your squid are so cute!” and guys. They’re right. I mean look at them:

It doesn’t get much more adorable than that, and I’m the first to admit it. But their cute-factor isn’t what gets me jazzed about the Hawaiian bobtail squid.What excites me is what these little squid have going on under the hood. Let me back up for a second and tell you why we study these squid in the first place.

Hawaiian bobtail squid are nocturnal predators on the reef, but much like many other squid, they are effectively swimming protein bars that bigger fish can’t wait to get their jaws on. Being in this position, many cephalopods have become extremely effective at hiding. Many of you may have already seen this video of octopus, but it’s a great demonstration of how well these animals do at hiding from the gaze of predators.

Octopus and cuttlefish have both become world class camouflagers, and Hawaiian bobtail squid have too, but instead of using color changing skin cells (chromatophores) to hide, the squid has developed a symbiosis with a bioluminescent bacterium called Vibrio fischeri. The squid keep these bacteria in a specialized “light organ” on their underside and are able to control the amount of light that escapes from the organ to match the moonlight coming from above.

Here’s a video of a closely related species of bacteria, Photobacterium leiognathi:

When squid first hatch, they need to find this bioluminescent bacterium swimming in the seawater. They’re extremely efficient at finding their partner, but what’s even more extraordinary to me, is that their immune cells can tell the difference between V. fischeri and other types of bacteria. This is totally nuts because compared to the human immune system, the squid immune system isn’t super complicated. In fact as far as we know, squid don’t have any kind of adaptive immunity or immunological memory at all. What we’ve noticed studying these immune cells (called hemocytes) is that not only do they treat V. fischeri differently, but the V. fischeri needs to be present in the light organ in order to maintain this tolerant hemocyte behavior. Previous work used antibiotics to remove V. fischeri from the squid, and after the treatment, squid immune cells no longer tolerated V. fischeri. So it seems that the bacterium is communicating, “Don’t eat me!”, to the immune system constantly.

The goal of my work is to understand how V. fischeri communicates with hemocytes to identify itself, and what hemocytes change about themselves and their behavior to accommodate for living in a symbiosis with V. fischeri. I’m exploring this by watching immune cells and how they interact with bacteria when I either raise squid with or without Vibrio fischeri. I’ve also compared how they treat various mutants of V. fischeri in an attempt to figure out what it is on the surface of V. fischeri or released from V. fischeri that’s the identifying factor. To do this, I bleed squid and then isolate the hemocytes from their blood (don’t worry, they survive the quick blood draw and are totally anesthetized when I draw blood from them).

Squid blood is blue!

I then label hemocytes with a live-cell stain called Cell Tracker Deep Red. This allows me to see my hemocytes (pink), and various species of bacteria (blue and yellow) all at the same time. I then visualize them on a confocal microscope. Confocal microscopes are particularly cool because they can focus on just one thin section of whatever you’re looking at, and then take pictures through multiple thin seconds. You end up with 3-D images, and for my work I end up taking 3D videos. Having beautiful videos is a definite bonus for working with this system.

This is what my data end up looking like! Hemocyte is pink, and two bacterial species are in blue and yellow.

In addition to my microscopy, our lab is also working on proteomics with squid immune cells. We’re hoping that by identifying the proteins that change between squid that are raised with and without V. fischeri, we’ll be able to determine what affects this change in hemocyte behavior after the addition of V. fischeri.

Squid are cute, hemocytes are cool, but is this important? (Spoiler: totally)

You might be asking yourself why any of this is important. Ok, your squid is cute, but why do we care what its immune cells are doing? Well, the thing about the human immune system, is that beneath all the bells and whistles of the adaptive immune system (antibodies, etc), we still have an innate immune system, which the squid has too. Partnering with bacteria is an ancient and widespread phenomenon in animals. Everything from the lowly hydra to a giraffe lives in association with bacteria (there are even efforts underway to characterize the microbiomes of all zoo animals which is pretty fun). If we learn how this squid can tell the difference between “good” and “bad” bacteria, we’re going to learn something about ourselves too. A lot of really nasty stuff happens when your gut microbiome gets out of whack, as you’ve probably heard. Messed up microbes are associated with many diseases and disorders, including diabetes, obesity, autism and MS. It’s really essential for us to understand how our immune systems are sensing who’s there, in order to see how these associations go wrong. We use the squid because the symbiosis with V. fischeri is super convenient, since the light organ only houses V. fischeri. Compare that to a mouse that can house up to 1000 species of bacteria. That’s a lot of noise to work though, so having one bacterial partner, especially one that you can raise squid without if you want to, makes for a great animal model.

If you want to learn more, there’s plenty of information out there about squid and this symbiosis.